EP3574086A1 - Method for reprogramming ruminant somatic cells - Google Patents

Abstract

The present invention relates to a method for in vitro preparation of stem cells from ruminant somatic cells, by the exogenous expression of genes CDX-2 and c-MYC in said somatic cells. The invention also relates to the stem cells thus obtained, as well as to the use thereof for expanding an individual genetic background, due to the amplification of the gene pool of the initial somatic cells, for screening anti-proliferation molecules and testing the action of anti-proliferation molecules, and for replicating and testing the virulence of certain pathogens. These stem cells have some of the features of pluripotent stem cells, such as self-renewal, infinite proliferation, the detection of telomerase activity, a specific cell cycle and the expression of particular markers.

Description

 PROCESS FOR REPROGRAMMING SOMATIC CELLS OF RUMINANTS

FIELD OF THE INVENTION

 The present invention relates to a method for the in vitro preparation of stem cells from ruminant somatic cells by the exogenous expression of the CDX2 and c-MYC genes in said somatic cells. The invention also relates to the stem cells thus obtained, as well as their use for the expansion of an individual genetic background, due to the amplification of the initial somatic cell gene pool, for the screening of anti-proliferative molecules and the test the action of anti-proliferative molecules, and to replicate and test the virulence of certain pathogens. These stem cells have certain characteristics of pluripotent stem cells, such as self-renewal, infinite proliferation, detection of telomerase activity, a specific cell cycle and the expression of particular markers. STATE OF THE ART

 Ruminants and in particular cattle represent a large part of the animal production in France, in Europe and in the world. Since the life cycle of animals is particularly long, the development of any method that will allow the long-term maintenance and amplification of an animal's genetic potential, via a cell with germinal potential in particular, is a priority objective for the subsectors. .

In particular, stem cells and embryonic stem cells (ES) have the unique characteristics of self-renewal and differentiation in vitro and in vivo in all embryonic lineages. For ES cells alone, there is colonization of the germ line with the property of producing descendants with the same genotype as that of the cells when they are injected into a recipient embryo. If ES cells have been isolated in several species, this strict definition of germinal colonization is currently restricted to rodents only. Other so-called ES-like cell types can be described in the absence of this fundamental property, especially for mammalian species other than rodents and non-mammalian species. These ES-like cells have been isolated, amplified and established in lines with self-renewing and differentiating properties in many species, including, but not limited to, humans, non-human primates, cash crops such as ruminants, sheep, goats, horses, rabbits, dogs and cats, birds, chicken and duck, fish like medaka, zebrafish, turbo, etc. Most of these cells have been characterized by their proliferation potential, in vitro differentiation and by the presence of certain markers such as surface antigens including SSEA1, SSEA3 and SSEA4, antigens initially identified in mice, but whose cross-reactivity with other species proved very important for identifying these cells (Koh and Piedrahita, 2014). Among these cells, very few have the property of colonization of the embryo, even at the somatic level with the notable exceptions of chicken cells (Pain et al., 1996) and zebrafish (zebrafish) (Hong et al., 2004 ).

 While ES-like cells are currently identified in many mammalian species, these cells are still particularly difficult to obtain from embryos and maintain in an undifferentiated state in long-term cultures. Many species are actually more or less refractory to the routine derivation of these cells (pigs, sheep, ruminants, dogs, cats, rabbits, etc.) and are inaccessible for specific genotypes or desired by the rarity and the number embryos generally available in these species (Koh and Piedrahita, 2014).

 In the last decade, somatic reprogramming has been one of the most promising discoveries in the field of stem cells. In 2006, Professor S. Yamanaka demonstrated for the first time that the introduction of a gene cocktail in a differentiated cell (a fibroblast) changed the fate of the cell by giving it the properties similar to those of embryonic stem cells ( ES). This reprogramming process was achieved by the combination of four transcription factors, the Oct4, Sox2, Klf4 and c-MYC gene products (OSKM), also called Yamanaka combination. Subsequently, various combinations of genes were also identified as the OSNL cocktail (Oct4, Sox2, Nanog and Lin28), called Thomson combination (Yu et al., 2007). Other actors, including Nr5a2 (Heng et al., 2009), ESRRB (Feng et al., 2009), ZIC3 (Declercq et al., 2013), TBX3 (Han et al., 2010), miR302, ( Anokye-Danso et al., 201 1) and others participate directly in the reprogramming process or increase reprogramming efficiency (Hochedlinger & Plath, 2009, Stadtfeld & Hochedlinger, 2010). But in any case, there is no major substitution in the original combination.

More recently, a process of somatic reprogramming called direct reprogramming has also been developed, which involves the use of a combinatorics of specific genes of a defined cell lineage to obtain, from a somatic cell, a lineage cell expressing reprogramming factors. It has thus been possible with an original combination of different transcription factors to generate functional cardiomyocytes with the combination Gata4, MEF2C, Tbx5 (Idea et al., 2010), with Ascii, Brn2, MytH to produce neurons (Vierbuchen et al. 2010), with Sox10, Olig2, ZPF536 to produce oligodendrocyte precursors (Yang et al., 2013) and with the combination FOXA3, HNF1A and HNF4A to produce hepatocytes (Yu et al., 2013, Huang et al. ., 2014). It is therefore through this process to obtain cells of a given lineage from the introduction into a differentiated somatic cell (such as a fibroblast) of a combination of transcription factors specific to the identified lineage.

 First demonstrated in the mouse model, the reprogramming approach was then successfully used on human fibroblasts, rat, non-human primate cells and with varying degrees of success in a large number of patients. mammals, including rabbits (Honda et al, 2013, Osteil et al, 2013), sheep and cattle. (Liu et al, 2012, Sumer et al, 201 1), pork (Ezashi et al, 2009), dog (Shimada et al, 2010) and even in some endangered species (Verma et al, 2013) . It is sometimes very difficult to check in the publications if the exogenous genes are no longer expressed and if the cells are therefore reprogrammed. In particular, the genetic stability of the cells presented is not illustrated and the question of their establishment over time remains open with the absence of a growth curve. In non-mammalian species, results are rarer with only a few controversial reports for avian cells (Lu et al 2012, Rosselo et al 2013) and tests on other species (Rosselo et al., 2013).

 In sheep and cattle, Sumer et al., 201 1 have described the production of induced pluripotent stem cells or "iPS" by modification of the conventional combination of OSKM genes to which the NANOG gene has been added. The authors demonstrate better efficacy with this addition and the expression of pluripotency markers such as alkaline phosphatase SSEA1 and SSEA4. The authors mention the obtaining of established cells or clones, without specifying the number of days or passages beyond 10 and 12. The only OSKM combination, without NANOG, only allows cells to be obtained from 7 to 9 passages. In both cases, there is no growth curve, and there is no long-term settlement.

Therefore, reprogramming attempts have always been made with the OSKM factors and / or with the variant associating the NANOG and LIN28 genes derived from the combinatorics of Thomson. In the literature, it seems difficult to obtain bovine cells that actually have the characteristics of stem cells or "ES-like", in particular on proliferation criteria, markers and in vitro differentiation potential in different embryonic lineages. .

SUMMARY OF THE INVENTION

 The present invention relates to a method for the in vitro preparation of stem cells, comprising ruminant somatic cell culture and exogenous expression of the CDX2 and c-MYC genes in said somatic cells.

 The CDX2 gene (caudal-type homeobox 2) is a caudal-type homeostasis gene that is a member of the transcription factor family that encodes a major regulatory protein for genes specifically involved in the differentiation and growth of gut cells. This protein plays a role in the early embryonic development of the intestinal tract.

 The invention also relates to ruminant stem cells as obtained by the above method (hereinafter referred to as BCM cells), and to their use for the expansion of an individual genetic background, because of the amplification of the initial somatic cell gene pool, for the screening of anti-proliferative molecules and the testing of the action of anti-proliferative molecules, and to replicate and test the virulence of pathogens.

 Preferably, and in addition to the expressed exogenous genes, these cells express telomerase (TERT) and in particular the following genes: GH1, LRP2, UGT8, JAG2, BRCA2, NBEA, CAMK2B, DSP, AMPH, SCIN, IGSF1, SLC6A15, CECR2 , and WNK2.

 The present invention also relates to the use of the exogenous expression of CDX-2 and c-MYC genes in somatic ruminant cells to prepare stem cells and various uses of ruminant stem cells obtained.

DETAILED DESCRIPTION OF THE INVENTION

 The present invention relates to a method for the in vitro preparation of stem cells, comprising ruminant somatic cell culture and exogenous expression of the CDX2 and c-MYC genes in said somatic cells.

The invention consists in using the CDX2 gene as the main reprogramming agent in association with c-MYC, in the presence or absence of pluripotency, such as OCT4 and / or SOX2 in particular, and germinal genes, such as DAZL, DDX4, NANOS and PIWI in particular. Taken independently, neither CDX2 nor c-MYC produced any effect on the different types of embryonic fibroblasts tested (from different ruminants), but in combination, BEF cells (Bovine Embryonic Fibroblast), GEF (Goat Embryonic Fibroblast), and SEF (Sheep Embryonic Fibroblast) show a drastic change in morphology a few days after the expression of combinatorics in cells. The cells obtained then have a morphology and kinetics of proliferation similar to those of embryonic stem cells.

 In the context of the invention, the genes mentioned are the following:

Table 1: * for the CDX2 genes, c-MYC, OCT4, SOX2, KLF4 of human origin and the NANOG and TERT genes of bovine origin, identified according to the NCBI database (https://www.ncbi.nlm.nih GENE search heading)

According to a preferred embodiment, the method for preparing cells according to the invention further comprises the exogenous expression of at least one of the genes selected from the group consisting of OCT4, SOX2 and KLF4, preferably at least OCT4, in said somatic cells. The invention thus makes it possible to obtain ruminant stem cells by reprogramming somatic ruminant cells. The cells that result from this reprogramming are unique in their characterization. They have similar properties to embryonic stem cells as self-renewal, rapid proliferation with an average doubling time between 10 and 13 hours against 60 hours for fibroblasts before reprogramming without phase of senescence, a typical cell cycle. a stem cell with a short G0 / G1 phase compared to that observed for fibroblasts, expression and activity of telomerase and the expression of a single molecular program.

 By "ruminant" is meant a group of unguligrade mammals that have the particularity of ruminating, namely the ability to remake their food (mainly grass and vegetables) after having first ingested them. There are three main ruminant families, the Bovidae, a family that includes ruminants with hollow horns and most domestic ruminants (beef, buffalo, goat, sheep, etc.), Giraffidae, short-horned and full-horned and Elaphids or Cervidae. , which includes ruminants whose horns fall annually when they have finished their growth (deer, fallow deer, reindeer, etc.). One of the physiological peculiarities of this group is the complexity of their stomach consisting of four distinct pockets allowing the rumination process.

 The method according to the invention has as "original product", or starting cells or initial cells, somatic cells of ruminants. By "somatic cells" is meant cells of a given lineage, other than stem cells, pluripotent stem cells and germ cells.

 Among the somatic cells that can be used in the process according to the invention, there may be mentioned fibroblasts, muscle cells, white blood cells circulating such as monocytes, macrophages, lymphocytes, etc., adipocytes, hepatocytes, cells endothelial, etc.

 The somatic cells used may be of any ruminant species, in particular they may be somatic bovine, goat or ovine cells.

 By definition, somatic cells do not express the telomerase gene or pluripotency genes such as the NANOG marker (Chambers and Tomlinson, 2009).

Therefore, the stem cell preparation method according to the invention uses somatic cells that do not express either the telomerase gene or the NANOG gene. In the context of the method of the invention, any type of gene delivery system can be used to drive the exogenous expression of CDX2 and c-MYC. Examples of delivery systems that may be mentioned include integrative strategies, particularly those using retroviral constructs, for example, or non-integrative strategies using, for example, the direct transfection of RNA, expression plasmids such as episomal plasmids, adenoviral infection, Sendai virus, etc. Efficient means have also been developed with polycistronic transgene expression cassettes. Virus-free systems based on floxinated transposon vectors for subsequent excision of transgenes have been developed (Woltjen et al., 2009, Kaji et al., 2009). All of these developments are aimed at minimizing the adverse and uncontrolled effect of transgene integration on genome integrity and the long-term stability of reprogrammed cells. The choice to produce a genetically altered (GA) cell that has only a minor scar from its modification is a step forward from the production of a genetically modified (GM) cell that retains the change permanently.

 Advantageously, the gene delivery system, whether integrative or non-integrative, is an inducible system, which can therefore be controlled at leisure. Thus, according to a particular embodiment, the expression of the exogenous genes in the cell preparation method according to the invention is an inducible expression. For example, gene expression may be driven by inducible vectors whose expression may be controlled herein by an inducing compound such as doxycycline for example. Thus, according to a preferred embodiment of the method of the invention, the expression of the CDX2 and c-MYC genes is an inducible expression.

 In the context of the present invention, the genes expressed in the somatic cells of ruminants may be from any origin in terms of species, and in particular of a species different from ruminants. Preferably, the expressed exogenous genes may be of human origin, or else ruminants, in particular of bovine origin.

The somatic cells are cultured in a suitable medium. The "appropriate medium" refers in particular to a conventional culture medium, adapted to somatic cells of ruminants, and most often liquid. Those skilled in the specialist field of cell culture will be able to determine the appropriate medium (s) for growing the cells in vitro, under optimized conditions, especially in terms of acid base equilibrium of the culture medium, temperature and controlled concentration of C0 ₂ .

 In the context of the invention, when the expression system chosen is an inducible expression system, the inducible expression is preferably carried out transiently. In this case, the inducing agent is preferably "removed" from the culture by progressive reduction, leading to a gradual decrease in the expression of exogenous CDX-2 and c-MYC genes, and, where appropriate, of the other gene (s). exogenous such as OCT4, SOX2 and KLF4. For example, in the case of induction with doxycycline, the concentration added to the medium is progressively reduced to result in its complete absence.

 The different exogenous somatic cell and expression cell culture steps of CDX-2 and c-MYC are performed for the time required. In particular, those skilled in the art will monitor the morphological change and reprogramming of the cells by visualizing, using a microscope, their growth and viability, and will be able to decide whether the step should continue beyond that. beyond the minimum time indicated.

 The somatic cell culture step is preferably carried out for about thirty days, at most about fifty days. In all cases, before entry into senescence of the cells.

 The exogenous expression step of the CDX-2 and c-MYC genes is preferably carried out over a period of at least 10 days, advantageously from 15 to 30 days, a period making it possible to stabilize the phenotype of the cells obtained. After this period, the induction of the expression of the CDX-2 and c-MYC exogenous genes, and, if appropriate, of the other exogenous gene (s) such as OCT4, SOX2 and KLF4 can be progressively reduced.

 According to a preferred embodiment, the process for the in vitro preparation of stem cells according to the invention comprises, in addition to the exogenous expression of CDX-2 and c-MYC, the expression of at least one of the genes selected from the group. consisting of OCT4, SOX2 and KLF4, preferably at least OCT4, in said somatic cells.

The reprogramming of somatic cells by the exogenous expression of CDX-2 and c-MYC results in a drastic morphological change, the elongated appearance of a large fibroblastic cell with an abundant cytoplasm of the original cells disappearing in favor of a small cell very refractive round in which the nucleocytoplasmic ratio is very high with a very limited cytoplasm, and by the acquisition of markers and cell properties comparable to stem cells, in particular in terms of self-renewal, proliferation, cell cycle, expression and activity of telomerase. The resulting product of the process consists of ruminant stem cells which can be maintained with the same phenotype, without a phase of senescence, aging or exhaustion, for at least 20 passages, preferably at least 30 passages, more preferably at least 50 passages, which corresponds to at least 80 generations, preferably more than 100 to 120 generations in a suitable culture medium. These ruminant stem cells express telomerase and the corresponding activity, present a cell cycle with a very short G0 / G1 phase, proliferate rapidly and do not express the markers OCT4, SOX2, NANOG pluripotency characteristic of embryonic stem cells.

 According to a second aspect, the present invention therefore relates to ruminant stem cells that can be obtained by the reprogramming method as defined above. The ruminant stem cells according to the invention are here called BCM cells for (Bovine CDX2 c-MYC) because of the exogenous expression of at least CDX2 and c-MYC.

 By "pluripotent stem cell" is meant the property of a cell to differentiate in vitro and in vivo in all cell types of an embryo and an organism (Niwa, 2007). Naïve and induced pluripotency states have been characterized more recently by cell origin and maintenance status in vitro (Nichols and Smith, 2009). All of these cells have specific molecular and epigenetic characteristics as well as specific developmental properties, including the ability to colonize the germline for naive pluripotent cells (De Los Angeles et al., 2015). Among the criteria used to characterize a pluripotent stem cell, mention may be made of cell self-renewal, the expression of transcription factors such as OCT4, SOX2, NANOG, DNA methylation levels which are often hypomethylated, and telomerase activity. , a particular cell cycle profile with a short G0 / G1 phase compared to that of a somatic cell.

 The cells obtained according to the invention are small round or ovoid adherent cells, but which can also proliferate in suspension without support when they are cultivated in untreated boxes for the culture. They do not express the pluripotency markers OCT4, SOX2, KLF4 and NANOG as defined in the literature according to the current consensus. These cells are therefore ruminant stem cells and not pluripotent stem cells; they can therefore also be defined as non-pluripotent stem cells of ruminants.

In addition to the combinatorics of exogenous genes that is expressed in somatic cells to obtain the stem cells of the invention, namely exogenous expression of CDX-2 and c-MYC possibly associated with the expression of at least one of the genes selected from the group consisting of OCT4, SOX2 and KLF4, these cells in particular express TERT telomerase, and at least one of the genes following: GH1, LRP2, UGT8, JAG2, BRCA2, NBEA, CAMK2B, DSP, AMPH, SCIN, IGSF11, SLC6A15, CECR2, WNK2. More preferably, they express at least two, at least three, at least four, at least eight, at least nine, at least ten, at least eleven, at least eight, at least twelve, at least six. least thirteen or fourteen of these genes. Even more preferentially, they express all of these fourteen genes.

 In addition, the stem cells of the invention do not express the following pluripotency endogenous genes: OCT4, SOX2 and NANOG. On the other hand, they express the exogenous proteins which correspond to the combinatorics of genes used: at least CDX2 and c-MYC, and possibly OCT4, SOX2, KLF4.

 In the context of the invention, the genes mentioned are the following:

Gene * Gene NCBI ID SeqRef Version Gene Name

 CDX2 618679 NM_001206299.1 24-DEC-2015 caudal type homeobox 2

GH1 280804 NM_180996.1 24-APR-2016 growth hormone 1

 LDL receptor related

LRP2 100337021 XM_015462263.1 26-JAN-2016

 protein 2

 UDP glycosyltransferase

UGT8 281566 NM_001083635.1 24-APR-2016

 8

 jagged 2, transcript

JAG2 517405 XM_015468638.1 26-JAN-2016

 variant X1

 breast cancer 2,

BRCA2 507069 XM_002684277.4 26-JAN-2016

 transcript variant X1 neurobeachin, transcript

NBEA 525624 XM_015465645.1 26-JAN-2016

 X5 variant

 Calcium / calmodulin-

CAMK2B 525416 NM_001035357.2 29-JUL-2016 dependent protein

kinase II beta Gene * Gene NCBI ID SeqRef Version Gene Name

 DSP 514360 NM_001 192368.1 04-JAN-2015 desmoplakin

 AMPH 614722 NM_001098130.1 24-APR-2016 Amphiphysin

 SCIN 281478 NM_174177.2 24-APR-2016 scinderin

 immunoglobulin

 IGSF1 1 540003 NM_001076921.1 25-AUGUST-2016

 superfamily member 1 1 solute carrier family 6

SLC6A15 281952 NM_181023.2 25-AUGUST-2016 (neutral amino acid)

 carry), member 15 cat eye syndrome

CECR2 517202 XM_015463582.1 26-JAN-2016 chromosome region, candidate 2

 WNK deficient lysine

WNK2 506520 XM_005199274.3 26-JAN-2016 protein kinase 2,

 X3 variant transcript

Table 2: NCBI basis (https://www.ncbi.nlm.nih.gov/ GENE search topic)

The stem cells of the invention exhibit telomerase activity. These cells express the TERT gene whose level of expression of the transcript is correlated with the activity of telomerase. This activity is detectable under conditions similar to mouse ES cells, taken as a reference for stem cells and known to express a significant telomerase activity; activity that is absent in the initial somatic fibroblasts.

 The proliferation capacity of the stem cells of the invention is infinite as tested with proliferation observed for at least 150 consecutive days without modification of their proliferation rate when they are maintained under identical culture conditions and without a notable change in phenotype.

According to a preferred aspect of the invention, the stem cells obtained have a stable phenotype during at least 20 passages, preferably at least 30 passages, more preferably at least 50 passages, from their establishment. By establishment, we mean the obtaining of cells which propagate perennially with a sustained, regular and constant proliferation rate over a significant period of time, beyond the somatic cell entry phase, evaluated at about 100 days for bovine fibroblasts.

The term "passage" refers to the culturing of cells having reached a maximum occupancy rate of their support. The cells are detached from their support by rapid action of an enzyme or a protease enzyme cocktail (trypsin, pronase, dispase, collagenase) dissociating the cells between them and promoting their detachment from the extracellular matrix ("trypsinization" action ") And are diluted in culture medium before being seeded on a new support for a new culture of a few days. Classically, cells detached from their support are counted before being seeded at a certain ratio of cells / cm ² in culture dishes.

 The stem cells of the invention also have a stem cell-specific cell cycle profile, ie less than 35%, preferably less than 31% of the cells in the G0 / G1 phase.

 Preferably, the phenotypic characteristics of the cells according to the invention are more particularly the following:

^■ Morphology of small refractile round or ovoid cell type with significant nucleocytoplasmic ratio

^■ Telomerase expression and activity

 ■ Phase distribution of the particular cell cycle with a low G0 / G1 cell count (<35%) compared with somatic cells (> 80%)

^■ Non expression of endogenous pluripotency genes such as OCT4, SOX2 and NANOG

^■ Expression of GH1, LRP2, UGT8, JAG2, BRCA2, NBEA, CAMK2B, DSP, AMPH, SCIN, IGSF1 1, SLC6A15, CECR2 and WNK2 genes.

 When the stem cells of the invention are obtained by the exogenous expression of CDX-2, c-MYC and OCT4, they also preferably express at least one of the following genes: APLP2, BEX5, ADGRG2, B3GNT3, CIST1 , KCNK17, MYH14, ADCY1. More preferably, they express at least two, at least three, at least four, at least five, at least six, at least seven or eight of these genes. Even more preferentially, they express all of these eight genes.

In this context, the genes mentioned are the following: Gene * Gene NCBI ID SeqRef Version Gene Name

 14-April-

POU5F1 282316 NM_174580.2 POU class 5 homeobox 1

 2016

 amyloid beta precursor like

APLP2 785005 XM_005197314.3 26-JAN-2016

 protein 2, X3 variant transcript

26-Aug-

BEX5 516056 NM_001075735.1 brain expressed X-linked 5

 2016

 G protein-coupled adhesion

ADGRG2 100299135 XM_015470573.1 26-JAN-2016 receptor G2, variant transcript

 X2

 UDP-GlcNAc: betaGal beta- 1, 3-N-

B3GNT3 784997 NM_001098473.1 24-APR-2016

 acetylglucosaminyltransferase 3

 intestine-specific transcript 1

CIST1 506589 NM_001075271.1 04-JAN-2015

 protein

 potassium channel, two pore

18-Aug-

KCNK17 282264 NM_174558.2 domain subfamily K, member

 2016

 17

 myosin, heavy chain 14, non

MYH14 514212 XM_010823804.2 26-JAN-2016

 muscle, variant X3 transcript

18-Aug-

ADCY1 281601 NM_174229.2 adenylate cyclase 1 (brain)

 2016

Table 3: According to the NCBI database (https://www.ncbi.nlm.nih.gov/; GENE search rubric

According to a third aspect, the present invention also relates to the use of the exogenous expression of CDX-2 and c-MYC genes in somatic ruminant cells to prepare stem cells. In a manner similar to that described above in relation to the process for obtaining these stem cells, the use according to the invention may comprise, in addition to the expression of the CDX-2 and c-MYC genes, the additional exogenous expression of at least one gene selected from the group consisting of OCT4, SOX2 and KLF4, preferably at least OCT4, in said somatic cells.

 All the preferred embodiments and all the precisions indicated above in connection with the implementation of the method according to the invention apply mutatis mutandis to the aspect of the invention concerning the use.

 Finally, the present invention also relates to the use of the stem cells of the invention for the expansion of an individual genetic background, or the genomic identification of this genetic background. The stem cells of the invention can also be used to screen for anti-proliferative molecules and to test the action of anti-proliferative molecules, in particular of certain drugs used for the treatment of cancerous pathologies, but also to replicate and test virulence. of pathogens in ruminants, including pathogenic strains of bovine tuberculosis, or to study the mechanisms of infection and spread of these pathogens. Finally, these cells can also be used as part of a veterinary regeneration process with a particular application in animal reproduction because of the potential of these cells for differentiation into germ cells.

 DESCRIPTION OF THE FIGURES

 Figure 1. Growth curve of BEF cells indicating their entry into senescence. Figure 2. Morphology of BCM cells (MC and OMC) compared to BEF cells before reprogramming

 Figure 3. Proliferation curve of BCM (MC and OMC) and BEF cells

Figure 4. Telomerase activity in BCM (TM, OMC) and BEF cells

Figure 5. BCM cells proliferate under non-adherent conditions

Figure 6. OMC cells express the proteins CDX2, c-MYC and OCT4 in the absence (- Dox), in the presence (+ Dox) and after weaning in doxycyline (DoxO) EXAMPLES

 Example 1. Growth Curve of Primary Fibroblasts

The proliferative potential of embryonic fibroblasts was evaluated, these data not being surprisingly available in the literature. Primary cultures of fibroblasts bovine embryos (BEF - Bovine Embryonic Fibroblast) were performed from the culture of explants taken from fetuses 70 days. The cells that proliferate from these explants were dissociated and reseeded at a concentration of 10 ³ to ² × 10 ³ cells per cm ² . These cells have a typical morphology of fibroblasts with the appearance of an elongated cell, more or less flattened, with abundant cytoplasm. In order to establish their long-term proliferative potential, the cells were dissociated at confluence in the presence of trypsin according to a conventional fibroblast maintenance protocol and maintained until they entered senescence, a phenomenon observed by a slowdown in their potential. proliferative and morphological change with the appearance of giant cells with a cytoplasm that leads to the gradual cessation of proliferation and the disappearance of the culture. At each passage, 1x10 ^e dissociated cells were seeded in culture dish of 55 cm ² in medium called BEF medium composed of DMEM / F12 base supplemented with 10% fetal calf serum (FBS), 1% L -glutamine (2mM), 1000 U / mL penicillin, 1000 U / ml streptomycin and maintained in a 38.5 ° C incubator at 7.5% CO ₂ . The medium was changed every 2-3 days. When the cells arrive at confluence, they were rinsed with PBS and dissociated with 0.05% Trypsin-EDTA whose action was stopped by the addition of complete culture medium. After centrifugation at 400 g, the dissociated cells were taken up in complete medium, counted and reseeded as indicated. Growth curves are based on counts.

All the different samples taken on different breeds give similar results with regard to the proliferation potential. The curves shown in Figure 1, indicate entry into senescence (limited number of generations) observed from 40 to 60 generations depending on the races corresponding to the 15-20 ^th passage when the number of harvested cells becomes identical or even lower than the number of cells seeded after a culture time above the average of the previous passages, about 3-4 days between each dissociation during the active proliferation period. During this process of proliferation and senescence, the cells progressively change their morphology and take on a larger and flattened cell morphology with a cytoplasm that becomes heterogeneous.

The proliferation differential between the fibroblasts and the cells obtained by reprogramming can thus be easily observed. Example 2. Constructs of inducible transposon vectors

 The bovine genome is only partially annotated especially in comparison with the human genome, whose knowledge is much more important. By comparing the coding phases of the human and bovine genes OCT4, SOX2, KLF4, c-MYC, NANOG and CDX2, it appears that the identities are very close (Table 4). The constructs were thus made with the human coding phases.

Table 4: Identity of the bovine-human sequences of the genes used

Human cDNAs were inserted into the inducible pPB vectors (Glover et al., 2013). All constructs were obtained with Gibson (NEB) technology using cloning oligonucleotides (Table 5) allowing the cloning of the coding phases.

The insertion of the coding phases was verified by sequencing. Genes Oligonucleotide sense (5 '>3') Antisense oligonucleotide (5 '>3') hOCT4 CcactagtcgagttaattATGGCGGGACAC gatcagttatctagattaatTCAGTTTGAATG

 CTGGCT (SEQ ID NO: 1) CATGGGAGAG (SEQ ID NO: 2) hSOX2 ccactagtcgagttaattATGTACAACATGA gatcagttatctagattaatTCACATGTGTGA

 TGGAGACG (SEQ ID NO: 3) GAGGGG (SEQ ID NO: 4) hKLF4 ccactagtcgagttaattATGAGGCAGCCAC gatcagttatctagattaatTTAAAAATGCCT

 CTGGC (SEQ ID NO: 5) CTTCATGTGTAAG G (SEQ ID NO: 6) hc-MYC ccactagtcgagttaattATGCCCCTCAACG gatcagttatctagattaatTTACGCACAAGA

 TTAGC (SEQ ID NO: 7) GTTCCG (SEQ ID NO: 8) ccactagtcgagttaattATGTACGTGAGCT gatcagttatctagattaatTCACTGGGTGAC hCDX2 ACCTCCTGG (SEQ ID NO: 9) GGTGGG (SEQ ID NO: 10)

Table 5: List of Oligonucleotides Used in Cloning Gene Coding Sequences in Expression Vectors Example 3. The OSKM Cocktail is Not Enough to Establish a Long-Term Proliferative Reprogrammed Cell in Cattle

Various reprogramming tests were conducted on the BEFs (see Example 1) at early passages (<passage 10). In tests with transposon vectors, the BEF cells were modified by electroporation. After dissociation, the cells were centrifuged at 1200 rpm (300 g) at room temperature for 5 min, after aspiration of the supernatant, the cell pellet was rinsed in PBS, centrifuged again and 1x10 ^e cells directly taken up in 120 μl of resuspension buffer ( Neon, Life Technologies). In this mixture was added in the form of a purified plasmid, 2 μg of transposase (1/3 of the total amount of plasmids) and 4 μg of vectors (2/3 of the total amount of plasmids), a mixture composed of the different inducible Piggybac transposons. doxycycline, the composition of which varies according to the combinations tested. The cell-plasmid mixture was electroporated by the Neon system (Life Technologies) at 1200V for 40ms in a 100μί cone dipped into a vessel containing electroporation buffer (Neon, Life Technologies). After electroporation, the cells were cultured in a 6-well plate well in 3 mL of complete fibroblast medium.

The medium of the electroporated cells was changed after 24 hours and selection by puromycin at 1 g / ml and by neomycin at 250 μg / ml was carried out according to the resistance genes carried by the plasmids present in the combinatorium. The selection medium was changed every other day for at least one week. At the end of selection between 8 and 12 days, the cells were dissociated by 0.05% trypsin-EDTA (Life) and 10 ⁵ cells were seeded in a 6-well plate well in 3 ml of medium. Two culture media well known to those skilled in the field of cell reprogramming have been tested: The "Stem" medium related to an EpiSC medium as defined in the mouse model is an aseric medium composed of DMEM / F12 ( 250 ml), supplemented with Neurobasal medium (250 ml), B27 supplements (5 ml), N2 supplements (2.5 ml), 2mM L-Glutamine, 1000 U / ml penicillin, 1000 U / ml streptomycin and of 1 mM β-mercaptoethanol. To this medium are added 5 ng / ml of b-FGF and 5 ng / ml of human Activin A (Peprotech), in the presence of doxycycline at 2 μg / ml final (Sigma).

The "ES" medium is composed of DMEM / F12 (500 mL) supplemented with 10% FBS, 2mM L-Glutamine, 1000 U / mL penicillin, 1000 U / mL streptomycin and 1 mM β-mercaptoethanol, 100 nM Sodium Pyruvate, 1% Non-Essential Amino Acid. The LIF factor (1000 U: mL) is added in the presence of doxycycline at 2 μg / mL final. In the "Stem" medium, morphological changes appear on average from 4 to 5 days after the addition of doxycycline while it is necessary to wait at least 7 to 10 days for the medium "ES". These changes are dependent on the presence of different coding phases present in the combinatorics.

The colonies of modified cells were removed after 10-12 days of induction and seeded in wells of 24-well plate previously treated in a poly-L-ornithine-laminin mixture (Sigma) allowing better adhesion. The proliferation and morphology of cells is very different from that of BEF fibroblasts and uninduced cells maintained in the absence of Doxycycline. But after an initial proliferative and morphological change, the modified cells gradually resume a fibroblastic morphology after several passages and can not establish in the long term. This is consistent with published observations (Sumer et al., 201 1). Example 4. The CDX2 gene induces a change in association with c-MYC

 Somatic cells derived from embryonic biopsies (see Example 1) were cultured until confluent. They were then dissociated and transduced to allow expression of the expression vectors of the inducible transgenes in the BEF medium (see Examples 2 and 3).

After the selection of the modified cells, they are then placed in the Stem medium and induced in the presence of Doxycyline. Morphological changes occur as early as 4 ^th or 5 ^th day after initiation of induction and stably remain from 8 to 10 days after the onset of induction. These cells are then passed and amplified and keep their newly acquired characters through this process.

 The single expression of CDX2 in BEF cells alone does not induce any significant morphological or proliferative changes compared to unmodified BEF. In the same way, the mere presence of c-MYC does not induce a major change in morphology and a slightly larger proliferation. But the combined expression of CDX2 and c-MYC induces a major morphological change of cells, as shown in Figure 2. Cells that express exogenous CDX2 and c-MYC genes (MC cells) and cells that express exogenous genes CDX2, c-MYC and OCT4 (OMC cells) become very small, round or ovoid and highly refractive with a very high nucleocytoplasmic ratio unlike fibroblast cells.

The presence of other pluripotency genes such as OCT4, SOX2 and KLF4 in the reprogramming cocktail does not significantly alter the kinetics of appearance of BCM cell colonies or their morphology.

Example 5. BCM cells are obtained in different breeds of ruminants

 Fibroblasts of Abondance, Holstein or meat breed origin were transduced under the described conditions with different combinations. After selection, the induction process of the transgenes was triggered by the addition of oxycyline in the culture medium and the morphological changes were observed after 5 to 7 days of induction with a slightly longer delay and a magnitude a little less for the meat breed (Table 6).

There is no major morphological difference in the cells obtained. Race

 Abundance Hollstein Meat Breed

 Obtaining reprogrammed cells

 combinatorics

0 - ^•

 i - iC ++ ++

 OiC +++ +++

 0SK1 -

Table 6

Example 6. BCM cells have stem cell characteristics

Indefinite proliferation

 The BCM cells, obtained in a reprogramming process involving at least the CDX2 and c-MYC combinatorics were dissociated at each passage, counted and reseeded in the presence of doxycycline and factors. Depending on the combinations used (MC or OMC), the proliferation curves make it possible to calculate an average doubling time for cells that proliferate in the absence of an inductor (FIG. 3). It is between 12.4 h and 15h for BCM cells from the MC combination (Table 7).

Table 7: Average Doubling Time of the Cells Obtained in Different Combinations

In addition, the proliferative potential of these cells is also related to the absence of senescence, unlike the initial BEF cells. Telomerase activity

 Telomerase activity was measured in BCM cells in comparison with BEF, using the TeloTAGGG Telomerase PCR ELISA commercial detection kit (Roche).

The cells were lysed and the telomerase activity was assessed by specific amplification of telomeric repeats (TRAP) followed by an ELISA as recommended by the manufacturer. The positive control (Ctrl +) is provided by the manufacturer as a witness to an important activity of telomerase.

 The BCM cells show a 90 and 170-fold higher activity compared to the BEF negative cells, respectively for the MC cells and the OMC cells (FIG. 4).

Specific cell cycle

 BCM cells also have a particular cell cycle profile. Proliferating cells in routine media were fixed and their average level of DNA was assessed by propidium iodide staining according to a standard protocol as described (Coronado et al, 2013). The cells were dissociated, counted, washed twice and then fixed with 70% ethanol at -20 ° C. for 2h minimum. The cell pellet was then resuspended in 5 ml of PBS and rehydrated by washing with PBS several times and then incubated for 30 min in PBS supplemented with 100 μg / ml of RNAse. The cells were analyzed by FACS (Becton) after addition of 50 μg / ml of propidium iodide by a Yellow Green laser (PE TexasRed). Cycles were run with 50,000 events and analyzed using the FlowJo analysis software to obtain the percentages of the different phases.

The cell cycle profile of OMC vs BEF cells is as follows:

 % G0 / G1 26.9 vs 83.9

 % S 52.6 vs 6.9

 % G2 10.3 vs 6.5

The cell cycle profile of MC vs BEF cells is as follows:

 % G0 / G1 30.4 vs 83.9

 % S 50 vs 6.9

% G2 1 1, 3 vs 6.5 The observed cell cycle data for MC and OMC cells, induced by the reprogramming method according to the invention, are typical of a stem cell and in high proliferation.

 The characteristics of the cells obtained according to the invention correspond well to characteristics of stem cells by their self-renewal, their telomerase activity, and their cell cycle profile.

Example 7. BCM cells proliferate in non-adherent condition

 After obtaining the BCM cells, the cultures are stabilized by the presence of a treatment in the culture well that allows them to be maintained, amplified and propagated. BCM cells proliferate in an adherent form in wells treated with poly-ornithine laminin and are preferentially non-adherent in the absence of treatment or even when the wells are treated with gelatin or fibronectin.

 These cells can be maintained in the form of small, non-adherent, proliferating aggregates in non-adherent cell culture flasks (Figure 5).

 The BCM cells of the invention are therefore able to proliferate both in adherent (Figure 2) and non-adherent (Figure 5) conditions.

Example 8. BCM cells express exogenous c-MYC, OCT4 and CDX2 proteins By Western blot analyzes, it is possible to study the expression of exogenous proteins OCT4, CDX2 and c-MYC with antibodies directed against proteins. human.

For this, the cells were lysed in a RIPA buffer (150mM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50mM Tris, pH 8, and protease inhibitor cocktail), incubated 30min in ice , and centrifuged. The supernatant containing the proteins was assayed (Coomassie Plus Bradford Assay Kit) at 595nm. 1 g of proteins were deposited and migrated on 12% acrylamide gel in 250mM Trizma buffer, 1900mM Glycine and 1% SDS, for 1 h 30 at 100 V, in the presence of a size marker (Biorad Precision Plus Protein). WesternC Standards # 161 -0376). The proteins were transferred to nitrocellulose membrane in 25mM Trizma buffer, 190mM Glycine and 20% methanol for 1 h at 100V. The membrane was then incubated in blocking buffer (TBS-Tween 5% milk) for 1 h and then with one of the following antibodies, in blocking buffer, overnight at 4 ° C or 1 h at room temperature: Antibody Origin Reference Use concentration

 Actin HRP Mouse A3854 sigma 1/10 000 - 5000

 Oct4 Rabbit sc-9081 1/500

 Cdx2 Rabbit sc-134468 1/500

 Myc Rabbit sc-788 1/500

 Table 8: Antibodies Used to Detect Exogenous Proteins

 The membrane was then washed 4 times 5min in TBS-Tween and then incubated with the secondary antibody, in blocking buffer for 1 hour at room temperature: Anti-rabbit HRP NA 934VS ECL Peroxidase 1/10 000. After 4 washes, the proteins have been revealed with Clarity ECL Substrate.

 As shown in FIG. 6, the OMC cells obtained using the combinatorial CDX2, c-MYC and OCT4 express the corresponding exogenous proteins in the presence of doxycycline (+ Dox) and maintain their expression after withdrawal of the cells into doxycycline ( Doxo).

Example 9. The minimal combinatorial CDX2 / C-MYC induces reprogramming in species other than cattle

 In order to test the effect of the CDX2 gene and its association with c-MYC in combinatorics, reprogramming was tested on mammalian embryonic fibroblasts, cells derived from 70-day-old bovine embryos, embryo fibroblasts 30 to 740 days of sheep and goat.

 All cells were transduced by the expression vectors with the human coding phases (bovine, sheep, goat).

 The important morphological changes have been observed for cattle, sheep, and goat cells with the possibility of obtaining cells that are established and have very similar morphologies to BCM cells (Table 9, the "+" symbol is in correspondence with the importance of morphological changes corresponding to those of stem cells).

 No significant changes are observed with murine or avian cells.

Combinatorial BEF SEF GEF

 VS - - -

M - - -

MC ++ ++ ++

WTO ++++ ++++ ++++ Table 9: Morphological changes observed with the CDX2 and c-MYC genes alone or in combination, possibly combined with OCT4.

Example 10. RNA Analysis of BCM Cells

 Seq RNA analysis was performed on BEF, MC and OMC cells. 1 Dg of

Total RNA extracted cells was used to prepare the library using the TruSeq R Stranded mRNA sample preparation kit (Illumina) according to the manufacturer's recommendation. The average fragment size estimated by Bioanalyser (Agilent) was 281 and 280 bp for the two BEF samples, 280 and 282 for the 2 samples of MC cells and 281 for the OMC cells. The molarity, estimated by Qubit (Life Technologies) was 250, 19 and 253.79 nM for BEF, 243.93 and 256.56 nM for MC cells and 236.71 nM for WTO cells. Each library is diluted to 10 nM in 10 mM Tris-HCl, 0.1% Tween 20. The samples are mixed, diluted to 4 nM, denatured in 0.2 N NaOH solution at room temperature for 5 min and then neutralized. by the addition of 200 mM TrisHCI pH 7. A dilution at a concentration of 20 μM is prepared in hybridization buffer. Paired end sequencing is performed on the NextSeq 500 sequencer (Illumina) using the 'nextSeq500 high output v2 (illumina) kit and sequencing quality controlled by the Sequencing Analysis Viewer 1.8 visualization software. 37 (Illumina). Quality control of the 'Sample-ID.fastq' files is done with the FasQC software (Babraham Institute). The total number of readings was 54362226 and 587473448 for BEF, 60,075,319 and 37,028,949 for MC cells and 90,334,233 for WTO cells. Sequence alignment was performed on the bovine genome, version Btau 5.0.1 https://www.ncbi.nlm.nih.gOv/assembly/GCA_000003205.6) using the Hisat, subread and R package (DEseq2) (Love et al., 2014).

Among the most differentially expressed genes between the BEF cells and the BCM (MC and OMC) cells, the genes GH1, LRP2, UGT8, JAG2, BRCA2, NBEA, are notably found, in addition to CDX2 and, where appropriate, OCT4, as exogenous genes. CAMK2B, DSP, AMPH, SCIN, IGSF1, SLC6A15, CECR2, WNK2. The results are summarized in Table 10 below. "Fold" Fold

 change "change"

 Gene * Gene NCBI ID SeqRef Version Gene Name

 MC vs OMC vs

 BEF BEF

 24-DEC- caudal type

CDX2 618679 NM_001206299.1 11, 45 10.63

 2015 homeobox 2

24-AVR

GH1 280804 NM_180996.1 11, 21 10.45 growth hormone 1

 2016

 26-JAN-LDL receptor

LRP2 100337021 XM_015462263.1 10.55 9.61

 2016 related protein 2

 UDP

 24-AVR

UGT8 281566 NM_001083635.1 10.54 10.39 glycosyltransferase

 2016

 8

 26-JAN-jagged 2, transcript

JAG2 517405 XM_015468638.1 10.13 10.40

 2016 variant X1

26-JAN- breast cancer 2,

BRCA2 507069 XM_002684277.4 10.08 9.29

 2016 transcript variant X1

26-JAN-neurobeachin,

NBEA 525624 XM_015465645.1 10.00 9.41

 2016 transcript variant X5 calcium / calmodulin-

29-JUIL-

CAMK2B 525416 NM_001035357.2 9.79 10.26 dependent protein

 2016

 kinase II beta

04-Jan-

DSP 514360 NM_001192368.1 9.65 10.12 Desmoplakin

 2015

 24-AVR

AMPH 614722 NM_001098130.1 9.57 9.15 amphiphysin

 2016

 24-AVR

SCIN 281478 NM_174177.2 9.38 9.26 scinderin

 2016

 immunoglobulin

25-Aug-

IGSF1 1 540003 NM_001076921 .1 9,22 8,99 superfamily member

 2016

 11 carrier carrier solute

25- AUG- 6 (neutral amino

SLC6A15 281952 NM_181023.2 9.18 7.87

 2016 acid transporter), member 15 cat eye syndrome

26-Jan-

CECR2 517202 XM_015463582.1 9.07 8.45 chromosome

 2016

 region candidate 2

WNK lysine

26-JAN- deficient protein

WNK2 506520 XM_005199274.3 9.03 8.53

 2016 kinase 2, variant X3 transcript

Table 10: NCBI-based (https://www.ncbi.nlm.nih.aov/; ru search brick GENE Among the genes differentially expressed between the MC and OMC cells, there are in particular, besides POU5F1 (= OCT4) as exogenous gene, the APLP2, BEX5, ADGRG2, B3GNT3, CIST1, KCNK17, MYH14 and ADCY1 genes. The results are summarized in Table 1 1 below.

Table 11: According to the NCBI database (https://www.ncbi.nlm.nih.gov/; conclusions

 The CDX2 gene has the potential to reprogram and allows in a minimal combination with c-MYC to obtain a new cell type, BCM cells. These BCM cells proliferate rapidly, indefinitely, have a telomerase activity and a corresponding specific cell cycle, and express different markers specific to stem cells.

 This minimal combinatorial CDX2 / c-MYC also works in sheep and goat cells. The stem cells obtained have very similar morphologies in all species.

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Claims

A method of in vitro preparation of stem cells, comprising ruminant somatic cell culture and exogenous expression of CDX-2 and c-MYC genes in said somatic cells.

 The method of preparing cells according to claim 1, further comprising exogenously expressing at least one of the genes selected from the group consisting of OCT4, SOX2 and KLF4, preferably at least OCT4, in said somatic cells.

 A method of preparing cells according to any one of the preceding claims, wherein the exogenous expression of the CDX-2 and c-MYC genes is carried out for at least 10 days.

 The method of preparing cells according to any one of the preceding claims, wherein the expression of said genes is an inducible expression.

 A method of preparing cells according to any one of the preceding claims, wherein said somatic cells are bovine, goat or ovine somatic cells.

 The method of preparing cells according to any one of the preceding claims, wherein said somatic cells are selected from the group consisting of fibroblasts, muscle cells, white cells of the circulating blood, adipocytes, hepatocytes and endothelial cells. .

7. Ruminant stem cells, obtainable by the method according to any one of claims 1 to 6.

 8. Ruminant stem cells according to claim 7, characterized in that they express the TERT telomerase gene and at least one of the following genes: GH1, LRP2, UGT8, JAG2, BRCA2, NBEA, CAMK2B, DSP, AMPH, SCIN, IGSF1, SLC6A15, CECR2, WNK2.

 9. Ruminant stem cells according to claim 8, characterized in that they exhibit a telomerase activity.

10. Ruminant stem cells according to claim 8 or claim 9, characterized in that they have a cell cycle profile with less than 35% of cells in G0 / G1 phase.

1 1. Using exogenous expression of CDX-2 and c-MYC genes in somatic ruminant cells to prepare stem cells.

 Use according to claim 11, characterized by the additional exogenous expression of at least one gene selected from the group consisting of OCT4, SOX2 and KLF4, preferably at least OCT4, in said somatic cells.

 13. Use of the stem cells according to any one of claims 7 to 10 for the expansion of an individual genetic background or the genomic identification of this genetic background, for screening anti-proliferative molecules and to test the action of molecules anti-proliferative agents, to replicate and test the virulence of pathogens in ruminants and to study the mechanisms of infection and spread of these pathogens.